Neisserial Opa protein dynamics and interactions with human CEACAM1

Neisseria gonorrhoeae and N. meningitides are human pathogens which infect millions each year and are becoming increasingly antibiotic resistant. These bacteria utilize opacity-associated (Opa) proteins to mediate bacterial uptake into non-phagocytic and phagocytic cells. Opa proteins primarily engage carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), hijacking host cellular mechanisms to induce bacterial engulfment. The Opa family of proteins are outer membrane β-barrels with eight transmembrane strands and four extracellular loops. The loops of Opa contain regions of high sequence variability that engage specific CEACAM receptors (termed hypervariable (HV) regions 1 and 2). Multiple sequence alignment of the HV regions does not reveal a conserved CEACAM-binding motif, and the molecular determinants of the Opa-CEACAM interaction are unknown.
Results from CW-EPR and previous NMR relaxation experiments indicate Opa loops are dynamic on the nanosecond timescale, which may enable a sampling of transient structures and loop interactions that promote receptor engagement. A combination of Double Electron Electron Resonance (DEER) and molecular dynamics (MD) were utilized to determine the unbound conformational ensemble of the dynamic Opa loops, where spin label pairs were selected as optimal MD restraints. Experimentally-restrained simulations of Opa show an association of the extracellular loops.
The N-terminal domain of CEACAM proteins (nCEACAM) dimerize in vivo and in vitro, which may impact Opa binding. Multiple crystal structures of the nCEACAM1 homodimer have been determined, so DEER distances of the dimer were acquired to refine the homodimer structure in solution. CEACAM proteins are also highly glycosylated in vivo, yet the role of these glycans on CEACAM oligomerization and interactions with Opa is unknown. Recombinant N-linked glycosylated nCEACAM1 proteins were generated using human cell cultures, and the addition of N-linked glycans did not alter the homodimerization propensity of nCEACAM1 proteins. N-glycosylated nCEACAM1 proteins are currently being used in Opa binding experiments to identify the effect of glycosylation on Opa interactions. Understanding the molecular mechanism of Opa-CEACAM binding will elucidate the manner by which pathogenic bacteria induce phagocytosis into human cells.